The fundamental mechanisms of DNA replication have been conserved throughout biology. The chemistry and direction of synthesis, the requirement for RNA primers, the mechanisms of semi-discontinuous replication with Okazaki fragments on the lagging strand and the need for well-defined origins are shared. The basic features of the replicative apparatus are also shared. All cells, both eukaryotic and prokaryotic, contain multiple DNA polymerases. Yet, only a subset of these polymerases can function as the replicative catalytic subunit.
In E. coli, Pol III holoenzyme is composed of ten different types of subunits that function in concert to perform highly rapid and processive DNA chain elongation from a primed template. The α subunit serves as the polymerization subunit; ε catalyzes a 3′-5′ exonuclease activity that is necessary for proofreading. θ binds to the N-terminal region of ε. Together, α, ε and θ associate tightly to form Pol III. The β subunit confers high processivity. It consists of a bracelet-shaped molecule that clamps around DNA, contacting the polymerase and preventing it from falling off of the template, ensuring high processivity. The asymmetric DnaX complex is responsible for transferring the sliding clamp onto a primer-terminus in an ATP-dependent reaction. The native holoenzyme appears to employ a DnaX complex containing two copies of the τ subunit and one copy of the shorter γ variant along with ancillary subunits (τ2γ1δδ′χψ). The dnaX gene expresses two related proteins; τ is the full length protein and γ is a truncated version formed by frameshifting during translation of dnaX. τ binds the α subunit of DNA polymerase III and causes it to dimerize, forming the scaffold upon which other auxiliary proteins can assemble to form a dimeric replicative complex. δ and δ′ are required for processive elongation in addition to their role in initiation complex formation. χ forms a 1:1 heterodimeric complex with ψ. χψ binds tightly to domain III of γ, while χ alone does not bind to γ. The interaction of ψ and γ is probably mediated through the conserved N-terminal region of ψ. χψ confers resistance to high salt on DNA synthesis catalyzed by holoenzyme, and this salt resistance requires the presence of SSB. χ interacts with C-terminus of SSB and enhances the binding of SSB to DNA, thereby preventing premature dissociation of SSB from the lagging strand and increasing holoenzyme processivity.
Bacterial DNA replication has long been recognized as an attractive target system for new antibacterials. It is an essential process and stalled DNA replication can trigger cell death. The bacterial DNA replication complex is target-rich and involves as much as 6% of the essential proteins in bacteria, and its proper functioning is based on multiple, dynamic enzyme-substrate, protein-protein, and protein-DNA interactions. Replication proteins tend to be highly conserved among bacteria but substantially different from eukaryotic systems at the amino acid sequence level, which may facilitate the identification of compounds that selectively disrupt bacterial DNA replication. With only a few copies per cell, the replication complex is a significant point of pathogen susceptibility and even very low concentrations of an inhibitor can shut down DNA replication.
Pseudomonas aeruginosa is a gram-negative bacterium is omnipresent in the environment in large part due to its propensity to grow on many different surfaces including tissues from plants and animals, rocks, soil as well as synthetic materials such as contact lens, surgical instruments and catheters. Pseudomonas aeruginosa causes a wide range of infections including bacteremia in urinary tract infections, burn victims and patients on respirators. In hospitals, Pseudomonas aeruginosa is responsible for about one-seventh of all infections with multidrug-resistant strains being increasingly common. The most serious medical problem caused by Pseudomonas aeruginosa are lung infections associated with cystic fibrosis (CF).
To date, published work relating to DNA replication in P. aeruginosa has focused on either characterization of the origin of replication or on the biophysical properties of the single-stranded binding protein (SSB).
Inhibition of bacterial DNA polymerase holoenzymes and other DNA replication-related processes will be beneficial in the treatment of bacterial infections especially against those organisms that have developed resistance to existing chemotherapeutics.